Process for producing nicotinamide adenine dinucleotide

ABSTRACT

THE PRESENT DISCLOSURE RELATES TO A METHOD FOR PRODUCING NICOTINAMIDE ADENINE DINUCLEOTIDE WHICH COMPRISES CULTURING A MICROORGANISM CAPABLE OF PRODUCING NICOTINAMIDE ADENINE DINUCLEOTIDE IN AN AQUEOUS NUTRIENT MEDIUM UNDER AEROBIC CONDITIONS IN THE PRESENCE OF AT LEAST ONE MATERIAL SELECTED FROM THE GROUP CONSISTING OF ADENINE ADENOSINE, ANDENOSINE MONOPHOSPHATE, ADENOSINE DIPHOSPHATE AND ADENOSINE TRIPHOSPHATE WITH OR WITHOUT A SECOND MATERIAL SELECTED FROM THE GROUP CONSISTING OF NICOTINIC ACID, NICOTINAMIDE, NICOTIN MONONUCLEOTIDE, NICOTINAMIDE MONONUCLEOTIDE, NICOTINIC ACID, RIBOSIDE, NICOTNIAMIDE RIBOSIDE, AND NICOTNINIC ACID ADENINE DINUCLEOTIDE. DERIVATIVES AND VARIOUS MIXTURES OF THESE COMPOUNDS MAY BE EMPLOYED.

United States Patent 3,709,786 PROCESS FOR PRODUCING NICOTINAMIDEADENINE DINUCLEOTIDE Kiyoshi Nakayama, Sagamihara-shi, Japan, assignorto Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan No Drawing. Continuation ofabandoned application Ser. No. 656,651, July 28, 1967. This applicationJune 17, 1968, Ser. No. 737,306

Claims priority, application Japan, July 29, 1966, 41/49,396, ll/49,397Int. Cl. C12d 13/06 US. Cl. 195-28 N Claims ABSTRACT OF THE DISCLOSUREThe present disclosure relates to a method for producing nicotinamideadenine dinucleotide which comprises culturmg a microorganism capable ofproducing nicotinamide adenine dinucleotide in an aqueous nutrientmedium under aerobic conditions in the presence of at least one materialselected from the group consisting of adenine adenosine, adenosinemonophosphate, adenosine diphosphate and adenosine triphosphate with orwithout a second material selected from the group consisting ofnicotinic acid, nicotinamide, nicotine mononucleotide, nicotinamidemononucleotide, nicotinic acid riboside, nicotinamide riboside, andnicotinic acid adenine dinucleotide. Derivatives and various mixtures ofthese compounds may be employed.

CROSS-REFERENCE TO RELATED APPLICATION This application is acontinuation of copending application Ser. No. 656,651, filed on July28, 196 7 and now abandoned.

The present invention relates to a process for producing nicotinamideadenine dinucleotide in high yields. More particularly, the presentinvention concerns a process for producing nicotinamide adeninedinucleotide by fermentation in the presence of at least one materialselected from the group consisting of adenine, adenosine, adenosinemonophosphate, adenosine diphosphate and adenosine triphosphate with orwithout at least one second material selected from the group consistingof nicotinic acid, nicotinamide, nicotine mononucleotide, nicotinamidemononucleotide, nicotinic acid riboside, nicotinamide riboside, andnicotinic acid adenine dinucleotide, including derivatives of all ofthese materials.

Nicotinamide adenine dinucleotide can be found in yeasts, molds orbacteria. Accordingly, a process for producing nicotinamide adeninedinucleotide by extracting the raw material from bacterial cells andpurifying the same is well known. yet, this process has itsdisadvantages.

Nicotinamide adenine dinucleotide is a compound having an important rolein biochemical reactions and is also useful in the alcoholicfermentation of glucose. Nicotinamide adenine dinucleotide, also knownas a coenzyme I, dehydrogenase I, diphosphopyridine-nucleotide orcozymase, has the following structural formula:

IIIHz Patented Jan. 9, 1973 "ice One of the objects of the presentinvention is to provide a process for producing nicotinamide adeninedinucleotide by a fermentation method on an industrial scale.

Another object of the present invention is to provide a process for thepreparation of nicotinamide adenine dinucleotide in high yields.

Other objects and further scope of applicability of the presentlnvention will become apparent from the detailed description givenhereinafter; it should be understood however, that the detaileddescription and specific examples, while indicating preferredembodiments of the present invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

Pursuant to the present invention, a process for producing nicotinamideadenine dinucleotide in high yields has been developed by adding to afermentation medium containing a microorganism capable of producingnicotinamide adenine dinucleotide at least one material selected fromthe group consisting of adenine, adenosine, adenosine monophosphate,adenosine diphosphate and adenosine triphosphate, with or without asecond material selected from the group consisting of nicotinic acid,nico tinamide, nicotine mononucleotide, nicotinamide mononucleotide,nicotinic acid riboside, nicotinamide riboside, nicotinic acid adeninedinucleotide and derivatives of these materials.

Heretofore, it has been known that nicotinamide adenine dinucleotidecontent may be increased by adding nicotinic acid or nicotinamide toanimal tissues or blood. However, the present inventors have found thatthe amount of nicotinamide adenine dinucleotide produced can beincreased by adding to the fermentation medium the adenine compoundslisted above, including the derivatives and substituted derivativesthereof, either alone or together with the nicotinic acid compoundslisted above, including the derivatives or substituted derivatives ofthese latter compounds.

The above-mentioned compounds may be added to the culture medium at anytime during the fermentation process. In the situation where both theadenine compounds and the nicotinic acid compounds are added to thefermentation medium, they may be added together or individually and,again, at any time during fermentation. When these compounds areproduced because of the specific character of the microorganismsemployed, the re sultant compounds effectively facilitate the productionof nicotinamide adenine dinucleotide.

Generally, the amount of the adenine compounds or adenine-nicotinic acidmixtures added to the fermentation medium will vary depending upon theparticular microorganisms utilized. Advantageously, the amount of thesecompounds added to the fermentation medium will vary from about 50mg./l. to about 10 g./l. Of course, these compounds may be added to theculture medium in the form of derivatives of these materials, forexample, in the form of appropriate salts, such as adeninehydrochloride, adenine sulfate, etc. The adenine and nicotinic acidcompounds used in the process of the present invention sometimes inhibitgrowth of microorganisms when high concentrations of these compounds areadded at the beginning of the cultivation. In this case, it ispreferable to add these compounds after the microorganism actuallygrows.

Any of the microorganisms which are capable of producing nicotinamideadenine dinucleotide can be used in the process of the presentinvention. Typical microorganisms include yeasts, bacteria, ray fungiand molds. Microorganisms which are particularly effective in increasingthe amount of nicotinamide adenine dinucleotide produced are yeastsbelonging to a genus such as Saccharomyces,

Candida, Torula, Schizosaccharomyces, Torulopsis, Hansenula, Endomyces,Rhodotorula, Zygosaccharomyces and so on, and bacteria belonging to thegenera Brevibacterium, Corynebacterium, Arthrobacter, Lactobacillus andStreptococcus. The microorganisms employed in the present invention arewidely distributed as shown in Table 1.

TABLE 1 Nicontinamide adenine dinucleotide accumulated in thefermentation liquor Microorganisms I II III IV V Aerobacter aeroaenesAICC 8308 :l: Aerobacter aerogertes ATCC 15247- i Agrobacteriamtamefactem ATCC 4720 :l: Ayrobacterium turnefacierts ATCC 4452. :l:Arthrobacter ureafacr'ens ATCC 7562 Arthrobacter citreus ATCC 11624- :i:Arthrobacter globtforrm's ATOC 8010.. i Arthrobacter tamescens ATGO 6947Azotobacter indicus ATCC 9037.... :i: Bacillus cere'as AICC 7004Brevibacterium acetylr'curn ATCC 9 Br iam n ATCO 68 Brevz'bacteriamammorrr'agenes ATGC 6872.- Brevibacteriurn helvolum ATCC 11822Brevibacteriam imperiale ATCC 8365 :b T Brevibacterr'um linens ATCC9175..-- Brevibacterium vitarurnen ATCC 10234.-. Corynebacteriurnmichiganese ATCC 10202- :i: C'oryrtcbacteriurn rathayi ATCC 13659Coryrtebacterium trr'tr'ci ATCC 11402. :1: Flavohacteriu'm arborescensAICC 4358. Staphylococcus epidermidis ATCC 155. Micrococcuslysodetkticus ATCC 4698- :1: Micrococcrts 80de7t8i8 AICC 15932 :1:Micrococcus varians ATCC 399 Pseudomonas aeruginosa ATOC 15246- :1:Pseudornonas putr'da ATCC 4359 :i: Pseudomonas boreopolis ATCC 15452..:b Proteus vulgaris ATOO 19181 i Serratr'a marcescerts ATCC 19180 :l:Sarcirta latea AICC 15176 :1: Streptococcus facealis ATCC 11420 -l-Xa'rtthomortas citri ATCC 15923 i Candida utilis ATCC 16321 1 Candidautt'lr's ATCC 9950 t .p Saccharomyces cerevisiae ATCC 15248....Saccharomyces cerevisiae ATOC 7754 :1: Saccharomyces carlsbergensis ATCC9080." Torula utr'lts ATOC 15239 A-+ Zygosaccharomyces major ATOC 15240.:1: -i- Candida tropicalis ATCC 15114 :i: Aspergillus niger NRRL (ATCC10254). :i:

Ustr'lago sphaerogerta ATCC 12421 :1: Pertr'ct'llium chrysogertum ATCC15421... :1: Streptomyces albus ATCC 618 :1: Streptomycea aureus ATCC3309 i Streptomyces antibioticas AICG 10382- :i: Streptomycesflavom'rens AICC 3320. :i: Streptomyces vinaceus NRRL-B 1381.- :1: .4-

NOTE.-I=N0 addition of adenine; II=100 ig/ml. of adenine added; III=300rig/ml. of adenosine triphosphate added; 1V=50 rig/ml. of adenine and100 llg./]1 'il. 0t nic0tinarnide added; V=150 [Lg-[1111. oi adenosinetriphosphate and 100 pgJml. of nicotine-made added.

The fermentation medium comprises either a synthetic culture medium or anatural nutrient medium which contains the essential nutrients for thegrowth of the microorganism strain employed. Such fermentation mediumgenerally contains a carbon source, such a carbohydrates, a nitrogensource, inorganic compounds and the like which are utilized by themicroorganism employed in specific amounts.

The carbohydrates include, for example, glucose, fructose, maltose,sucrose, starch, starch hydrolysate, molasses and the like. Smallamounts of other suitable carbon sources, such as glycerol, mannitol,sorbitol, organic acids, hydrocarbons, etc., may be used in thefermentation me dium along with the carbohydrates. The carbohydrates maybe used either singly or in mixtures of two or more and any small amountof other carbon sources may also be present either singly or in mixturesof two or more.

The inorganic compounds include such materials as potassium phosphate,magnesium sulfate, iron sulfate or other iron salts, potassium chloride,magnesium chloride, calcium chloride, etc. As a nitrogen source, variouskinds of inorganic or organic salts or compounds, such as urea orammonium salts such as ammonium chloride, ammonium sulfate, ammoniumnitrate, ammonium phosphate, etc., or one or more than one amino acidmixed in combination, or natural substances containing nitrogen, such ascornsteep liquor, yeast extract, meat extract, fish meal,

for example, aspartic acid, threonine, methionine, etc., and/orvitamins, for example, biotin, thiamine, cobalamin and the like.

Fermentation is conducted under aerobic conditions, such as aerobicshaking of the culture or with stirring of a submerged culture, anincubation temperature of about 20 to 40 C. and a pH of about 5 to 9being preferable. Remarkably large amounts of nicotinamide adeninedinucleotide are found to be accumulated in the fermentation liquor andmicrobial cells.

Other conditions of temperature and pH may also be used with loweryields.

After the completion of the fermentation, the nicotinamide adeninedinucleotide may be separated from the culture liquor by conventionalmeans, such as ion exchange resin treatment, precipitation with metallicsalts, extraction methods, conventional adsorption methods,chromatography and the like.

The following examples are given merely as illustrative of the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the percentages in the application and in the examples are byweight.

EXAMPLE 1 Corynebacteriurn sp. No. 3485, ATCC 21084, is used as the seedbacterium. It is cultured at 30 C. for 24 hours in a seed mediumconsisting of 2% glucose, 1% peptone,

1% yeast extract, 0.3% sodium chloride and 30 micrograms per liter ofbiotin.

The fermentation medium employed has the following composition:

50 g. glucose 6 g. urea 2 g. KH2PO4 g. yeast extract 30 micrograms ofbiotin per liter of the culture medium One milliliter of a 12% solutionof urea is separately sterilized and added to 19 ml. of the fermentationmedium which has been previously sterilized in an autoclave under theconditions of 1 kg./crn. for 10 minutes.

The seed culture is inoculated into the fermentation medium in an amountof 10% by volume thereof. The mixture of media is then poured in ml.portions into individual 250 ml. conical flasks. After sterilizationunder pressure, fermentation is carried out with aerobic shaking of theculture at 30 C. After 24 hours of culturing, adenine is added to thefermentation liquor to give a concentration of 2 mg./ml. and then thecultivation is continued for 72 hours. 210 micrograms per milliliter ofnicotinamide adenine dinucleotide is accumulated in the fermentationliquor. The nicotinamide adenine dinucleotide is recovered by an ionexchange resin treatment.

The same culture as mentioned above is carried out but without addingadenine after 24 hours of culturing. In this case, the amount ofnicotinamide adenine dinucleotide accumulated in the fermentation liquoris 37 micrograms per milliliter.

EXAMPLE 2 Culturing is carried out in the same manner and under the sameconditions as described in Example 1, except that adenosine is added,instead of adenine, to give a concentration of 2 mg./ml. in the culturemedium. The amount of nicotinamide adenine dinucleotide produced is 183micrograms per milliliter.

EXAMPLE 3 Culturing is conducted in the same manner and under the sameconditions as described in Example 1, except that 5-adenylic acid(adenosine monophosphate) is used instead of adenine. The amount ofnicotinamide adenine dinucleotide produced is 173 micrograms permilliliter.

EXAMPLE 4 This example follows the same procedure as Example 1, exceptthat adenosine diphosphate is used instead of adenine. The amount ofnicotinamide adenine dinucleotide produced is 172 micrograms permilliliter.

EXAMPLE 5 This example follows the same procedure as Example 1, exceptthat adenosine triphosphate is used instead of adenine. The amount ofnicotinamide adenine dinucleotide produced is 187 micrograms permilliliter.

EXAMPLE 6 Each yeast culture listed in Table 2, precultured overnight inKoji liquor of Baum 10 at 30 C., is inoculated into 50 ml. of Kojiliquor of Baum 10 in a 250 ml. conical flask in an amount of 1%, and astatic culture is conducted for 2 days. The amount of yeast cellsproduced and the nicotinamide adenine dinucleotide content in the yeastcells are compared in Table 2 below for the cases when 50 micrograms permilliliter of adenine is added and when adenine is not added to theculture medium at the time of inoculation.

Amount Nicotinamide of yeast adenine Yeasts cells 1 dinucleotide 1Caniiida utilis, ATCC 16321:

1 Dry weight, g./l. 2 Mg./dried yeast cells, 1 g. N 0'rE.-I=Adenine notadded; II=Adenlne added.

EXAMPLE 7 This example follows the same procedure as that of Example 1,except that Arthobacter sp. :No. 3486, ATCC 21085, is used instead ofCorynebacterium sp. No. 3485, ATCC 21084 as the microorganism. Also,adenosine triphosphate is employed instead of adenine. The amount ofnicotinamide adenine dinucleotide produced is 270 micrograms permilliliter.

In the case where no adenosine triphosphate is added to the medium, as acontrol, the amount of nicotinamide adenine dinucleotide accumulated is34 micrograms per milliliter.

EXAMPLE 8 Corynebacterium sp. No. 3485, ATCC 21084, is used as the seedbacterium. It is cultured at 30 C. for 24 hours in a seed mediumconsisting of 2% glucose, 1% peptone, 1% yeast extract, 0.3% sodiumchloride and 30 micrograms per liter of biotin.

The fermentation medium employed has the following composition:

50 g. glucose 6 g. urea 10 g. yeast extract 30 micrograms of biotin perliter of the medium One ml. of a 12% solution of urea is separatelysterilized and added to 19 ml. of the fermentation medium which has beenpreviously sterilized in an autoclave under the conditions of 1 kg./cm.for 10 minutes. The seed culture is inoculated into the fermentationmedium in an amount of 10% by volume thereof. The mixture of media isthen poured in 20 ml. portions into individual 250 m1. conical flasks.After sterilization under pressure, fermentation is carried out withaerobic shaking of the culture at 30 C. After 72 hours of culturing, thecompounds shown in Table 3 are added to the fermentation medium to givea concentration of 2 mg./ml. After an additional 48 hours of culturing,nicotinamide adenine dinucleotide is accumulated in the fermentationliquor and recovered by an ion exchange resin treatment. The amounts ofthe product recovered are shown in Table 3.

TABLE 3 Nicotinamide adenine Compounds added: dinucleotide, ,ugjml.

Nicotinic acid+adenine 510 Nicotinic acid-i-adenosine 520 Nicotinicacid+adenosine monophosphate 430 Nicotinic acid+adenosine diphosphate320 Nicotinic acid-l-adenosine triphosphate 330 Nicotinamide+adenine 610Nicotinamide+adenosine 570 Nicotinamide+adenosine monophosphate 430 7TABLE 3-Continued Nicotinamide adenine Compounds added: dinucleotide,,ug/ml.

Nicotinamide+adenosine diphosph-ate 350 Nicotinamide-l-adenosinetriphosphate 420 Nicotinic acid mononucleotide+adenine 410 Nicotinicacid mononucleotide-i-adenosine 420 Nicotinic acidmononucleotide+adenosine monophosphate 330 Nicotinic acidmononucleotide+adenosine diphosphate 370 Nicotinic acidmononucleotide+adenosine triphosphate 350 Nicotinamidemononucleotide+adenine 290 Nicotinamide mn0nucleotide+aden0sine 280Nicotinamide mononucleotide+adenosine monophosphate 250 Nicotinamidemononucleotide+adenosine diphosphate 250 Nicotinamidemononucleotide+adenosine triphosphate 250 Nicotinic acidriboside-l-adenine 480 Nicotinic acid riboside+adenosine 480 Nicotinicacid riboside+adenosine monophosphate 320 Nicotinic acidriboside-l-adenosine diphosphate 345 Nicotinic acid riboside+adenosinetriphosphate 345 Nicotinamide riboside+adenine 300 Nicotinamideriboside+adenosine 345 Nicotinamide riboside+adenosine monophosphate 370Nicotinamide riboside-i-adenosine diphosphate 370 Nicotinamideriboside+adenosine triphosphate 390 Nicotinic acid adeninedinucleotide-kadenine 320 Nicotinic acid adeninedinucleotide-i-adenosine 320 Nicotinic acid adeninedinucleotide+adenosine monophosphate 270 Nicotinic acid adeninedinucleotide-i-adenosine diphosphate 240 Nicotinic acid adeninedinucleotide+adenosine triphosphate 270 No addition 37 EXAMPLE 9 Thisexample follows the same procedure as Example 8, except thatArthrobacter sp. No. 3486, ATCC 21085, is employed as the microorganism.The amounts of nicotinamide adenine dinucleotide accumulated in thefermentation medium are shown in Table 4.

TABLE 4 Nicotinamide adenine Compounds added: dinucleotide, pg./ ml.

Nicotinic acid+adenine 530 Nicotinamide-l-ade-nine 480 Nicotinicacid-i-adenosine triphosphate 470 Nicotinamide+adenosine triphosphate550 No addition 34 EXAMPLE 10 Each yeast culture listed in Table 5,precultured overnight in a Koji liquor of Baume 10 at 30 C., isinoculated into 50 ml. of Koji liquor of Baume 10 in a 250 ml. flask inan amount of 1%, and a static culture is conducted. The amount of yeastcells produced and the nicotinamide adenine dinucleotide content in theyeast cells are compared in Table 5 below in the case when 100micrograms per milliliter of nicotinamide and adenine is added to thefermentation medium and in the case when these compounds are not addedto the medium 24 hours after inoculation.

Caniiida tilts, ATCC 16321:

0. 9 3. 2 IL 1.0 6. 4 Sacchromyces cerev ae, ATCC 152 1.0 0. 4 II 1. 25.1 Candida tropicalis, ATCC 15114:

1 Dry weight, g./l.

2 Mg./g., yeast cells.

Norn.I=Adenine and nicotinamide not added; II=Adenine and nicotinamideadded.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included herein.

I claim:

1. A process for producing nicotinamide adenine dinucleotide whichcomprises culturing a nicotinamide adenine dinucleotide-producingmicroorganism belonging to a genus selected from the group consisting ofAerobacter, Agrobacterium, Athrobacter, Azotobacter, Bacillus,Corynebacterium, Flavobacterium, Staphylococcus, Micrococcus,Pseudomonas, Proteus, Serratia, Sarcina, Streptococcus, Xanthomonas,Candida, Saccharomyces, Torula, Zygosaccharomycee, Aspergillus,Ustil-ago, Penicillium and Streptomyces or belonging to a genus-speciesselected from the group consisting of Brevz'bacteriumacetylicum,Brevibacterium helvolum, Brevibacterium imperiale, Brevibacterium linensand Brevibacterium vitariumen in an aqueous nutrient medium underaerobic conditions in the presence of an adenine compound selected fromthe group consisting of adenine, adenosine, adenosine monophosphate,adenosine diphosphate, adeosine triphosphate and mixtures thereof,accumulating the nicotinamide adenine dinucleotide in the resultantculture liquor and in the microbial cells and isolating the nicotinamideadenine dinucleotide therefrom.

2. The process of claim 1, wherein a nicotinic acid compound selectedfrom the group consisting of nicotinic acid, nicotinamide, nicotinemononucleotide, nicotinamide mononucleotide, nicotinic acid riboside,nicotinamide riboside, nicotinic acid adenine dinucleotide, and mixturesthereof is also added to said medium.

3. The process of claim 1, wherein from about 50 mg./ 1. to about 10g./l. of said adenine compound is present in the medium.

4. The process of claim 2, wherein from about 50 mg./ l. to about 10g./l .of the mixture of said adenine compound and said nicotinic acidcompound is present in the medium.

5. The process of claim 3, wherein culturing is carried out at atemperature of from about 20 to 40 C. and at a pH of from about 5 to 9.

6. The process of claim 4, wherein culturing is carried out at atemperature of from about 20 to 40 C. and at a pH of from about 5 to 9.

7. The process of claim 1, wherein said microorganism is a yeastbelonging to a genus selected from the group consisting ofSaccharomyces, Candida, Torula, Schizosaccharomyces, Torulopsis,Hansenula, Endomyces, Rhodotorula and Zygosaccharomyces.

8. The process of claim 2, wherein said microorganism is a yeastbelonging to a genus selected from the group consisting ofSaccharomyces, Candida, Torula, Schizosaccharomyces, Torulopsis,Hansenula, Endomyccs, Rhodotorula and Zygosaccharomyces.

9. A process for producing nicotinarnide adenine dinucleotide whichcomprises culturing a nicotinarnide adenine dinucleotide-producingmicroorganism belonging to a genus selected from the group consisting ofAerobacter, Agrobacterium, Arthrobacter, Azotobacter, Bacillus,Corynebacterium, Flavobacterium, Staphylococcus, Micrococcus,Pseudomonas, Proteus, Serratia, Sarcina, Streptococcus, Xanthomonas,Candida, Saccharomyces, Torula, Zygosaccharomyces, Aspergillus,Ustilago, Penicillum and Streptomyces or belonging to a genus-speciesselected from the group consisting of Brevbacterium acetylicum,Brevibacterium helvolum, Brevibacierium imperiale, Brevibacterium linensand Brevibacterium vitarumen in an aqueous nutrient medium under aerobicconditions at a temperature of from about 20 to 40 C. and at a pH offrom about 5 to 9 in the presence of an adenine compound selected fromthe group consisting of adenine, adenosine, adenosine monophosphate,adenosine diphosphate, adenosine triphosphate and mixtures thereof,accumulating the nicotinarnide adenine dinucleotide in the resultantculture liquor and in the microbial cells, and isolating thenicotinamide adenine dinucleotide therefrom.

10. The process of claim 9, wherein a nicotinic acid compound selectedfrom the group consisting of nicotinic acid, nicotinarnide, nicotinemononucleotide, nicotinarnide mononucleotide, nicotinic acid riboside,nicotinarnide riboside, nicotinic acid adenine dinucleotide and mixturesthereof is also added to said medium.

References Cited UNITED STATES PATENTS 3,368,947 2/1968 Nakayama et a1.19528 N ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 195-114

